Lectins play critical roles in the immune system. The lectins on dendritic cells participate in antigen recognition and internalization and thereby serve as critical mediators of immunity. One such lectin, DC-SIGN, which mediates the beneficial process of antigen uptake for processing, can be co-opted by pathogens to suppress immune responses and promote their dissemination. For example, DC-SIGN can bind to HIV and facilitate its dissemination. It also has been shown to suppress immune responses to Mycobacterium tuberculosis. Because HIV and Mycobacterium tuberculosis are the two most significant threats to human health worldwide, it is critical to elucidate the molecular mechanisms underlying the function of DC-SIGN. The long-term goals of the proposed research are to use chemical biology to illuminate how DC-SIGN functions in signaling, antigen recognition, and antigen internalization. Insight into these processes can facilitate the design of agents to enhance the effectiveness of vaccines or prevent pathogen infection. The specific aims of the project follow: (1) to identify non-carbohydrate, small-molecule ligands for DC- SIGN, (2) to develop chemical imaging strategies to follow DC-SIGN-mediated antigen internalization and dendritic cell migration, and (3) to develop new chemical approaches to explore bacteria-dendritic cell interactions mediated by DC-SIGN. The first specific aim focuses on providing a novel set of inhibitors of DC-SIGN. These also can serve as tools for exploring the consequences of engaging this lectin. The DC-SIGN ligands identified to date are based on carbohydrates, which bind weakly and can interact with other dendritic cell-surface lectins. We envision that non-carbohydrate-based ligands will bind to DC-SIGN with higher specificity and affinity. In Aim 2, we shall use the ligands identified in Aim 1 to explore the consequences of antigen interaction with DC-SIGN. DC-SIGN interacts with a variety of different types of antigens (proteins, viruses, bacteria, and fungi); therefore, it is critical to understand how antigen structure influences internalization and trafficking. We shall use chemical synthesis to vary DC-SIGN ligand structure and to endow these model antigens with fluorogenic reporter groups for imaging. Aim 3 addresses how dendritic cell internalization and processing of organisms is influenced by DC-SIGN engagement. To investigate this process, we shall chemically modify bacteria such that they display specific DC-SIGN ligands. The results of the investigations proposed in Aim 3 can provide insight into the role of DC-SIGN and offer new strategies to explore the role of specific receptors in host-pathogen interactions. Relevance. The carbohydrate-binding protein DC-SIGN is found on the surface of dendritic cells, where it functions in the immune system. In addition to its normal function in humans, DC-SIGN can be used by viruses, like HIV, to facilitate their dissemination, and by some bacteria, like those that cause tuberculosis, to suppress immune responses. Because of its role in facilitating the two diseases that cause the most deaths worldwide, how DC- SIGN works and how to control it is important. This project is aimed at identifying DC-SIGN inhibitors and using compounds that bind to DC-SIGN to study its function.